Abstract: The discovery of the anti-viral APOBEC3 proteins is regarded as one of the most therapeutically promising breakthroughs in HIV/AIDS molecular virology in recent years. Humans have seven APOBEC3 proteins and at least two, APOBEC3F and APOBEC3G, are known to inhibit the replication of Vif-deficient HIV. HIV pathogenesis is consequently dependent on its Vif protein, which counteracts the APOBEC3 proteins and triggers their degradation. This proposal has three major aims. First, we will test the hypothesis that at least three or more APOBEC3s contribute to HIV restriction in vivo. A combination of methods will be used to identify the minimal set of APOBEC3s required for restriction including gene targeting, short hairpin RNA knockdowns, experiments with APOBEC3-resistant virus isolates and the use of novel APOBEC3 inhibitors. These studies will show definitively which APOBEC3s contribute to HIV restriction in non-permissive T-cell lines and pathogenesis-relevant primary CD4+ T lymphocytes. Second, we will test the hypothesis that APOBEC3 proteins are regulated by phosphorylation. The residues responsible will be identified by a combination of mutagenesis and phospho-peptide mapping. Preliminary studies indicate that APOBEC3G is phosphorylated minimally at three distinct residues. Phosphorylation may function to hard-wire APOBEC3G to signal transduction pathways, allowing for rapid activation of the protein in response to infection. Third, we will test the hypothesis that the APOBEC3 proteins homo- and hetero-oligomerize to form a combinatorial restriction network with a broader range of anti-viral activities exceeding those of individual APOBEC3s. The higher order network will be elucidated and its relevance to restriction evaluated by co-immunoprecipitation, co-encapsidation, single cycle, and spreading infection assays. The interaction domains will be determined by complimentary yeast-based and structure-guided mutagenesis experiments. Taken together, these studies will advance our fundamental understanding of the HIV-relevant human APOBEC3 proteins and facilitate the development of novel HIV/AIDS therapeutics that work by modulating the APOBEC3-Vif pathway.